In the double antibody immunoassay reaction an amount of labelled antigen and unlabelled antigen compete to react with an amount of antibody that is insufficient to bind all of the labelled and unlabelled antigen. An equilibrium is set up in which the amount of bound labelled antigen and free labelled antigen is controlled by the amount of unlabelled antigen present in the assay. The amount of unlabelled antigen can then be determined by measuring the amount of free or bound labelled antigen and a calibration curve generated by the use of standard preparations of unlabelled antigen.
The technique is described, with examples, in a Review Paper by R. S. Yalow and S. A. Berson in IAEA-SM-124/106, pages 455-481.
Various techniques are known for separating the bound from the free antigen. One technique involves attaching the antibody to an insoluble carrier such as described in U.S. Pat. No. 3,853,987.
Another technique for separating the bound from the free antigen involves the precipitation of the antigen-antibody complex out of solution by the addition of a second antibody directed against the serum or gamma globulin of the animal species used to generate the first antibody. This second antibody may be added either in solution or adsorbed or covalently bound to an insoluble carrier such as cellulose.
Both types of second antibody addition suffer from disadvantages. Addition of the second antibody adsorbed on a solid carrier gives rise to difficulties in ensuring that homogeneous slurry additions are made, and slower equilibrium attainment is generally found. Additionally, quite a large amount of second antibody is generally required and relatively complex chemical manipulations (to prepare the insolubilized material) are needed, resulting in greatly increased cost.
Addition of the second antibody as a solution avoids most of these problems; but it can result in the formation of only a minute quantity of whitish or translucent precipitate which may be virtually invisible after centrifuging. Inability to see the solid deposit in the assay tube makes it difficult for an operator, particularly an inexperienced one, to remove the supernatant liquid before measurement of the activity of the deposit.
One solution to this problem which has been used is to add extra carrier serum, (serum from a nonimmunized animal of the same species in which the primary antiserum is made), while still using an increased amount of the second antibody, so as to increase the physical bulk of the precipitate. This solution, however, requires considerable second antibody and often results in the same translucent pellet that is difficult to handle because of poor visualization.
Another solution described in U.S. Pat. No. 3,985,867 involves coloring the second antibody so that the precipitate formed is colored and is thus more clearly visible. This procedure, however, involves a complex process for preparing the dyed antibody in which the second antibody must be coupled to a solid phase bearing the first antibody for dyeing, washed, and then separated from the solid phase. Assay procedures using the dyed second antibody require the same large volume of the antibody as do conventional assays, as well as nonimmune carrier serum, in order to obtain a pellet large enough to be visible. Further, binding a dye to the antibodies can inhibit their efficiency in the immunoassay.